IL-16 antagonist peptides and DNA encoding the peptides

ABSTRACT

The present invention has found that a series of peptides having sequences that substantially correspond to specific regions of the C-terminus of IL-16 can inhibit the activity of IL-16. The present invention has demonstrated that such IL-16-inhibiting peptides can be as short as 4 amino acid in length. Based on these discoveries, the present invention provides IL-16 antagonist peptides and the use thereof for the treatment of IL-16 mediated disorders such as certain inflammatory diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of Ser. No. 10/358,627, filedFeb. 5, 2003, which is a continuation of Ser. No. 09/368,630, filed Aug.5, 1999, now U.S. Pat. No. 6,699,466.

This invention was made in the course of work under grant HL32802sponsored in part by the National Institute of Health.

FIELD OF THE INVENTION

The present invention relates to IL-16 antagonists and the use thereoffor the treatment of IL-16 mediated disorders such as certaininflammatory diseases. In particular, the present invention relates tothe discovery of IL-16 antagonist peptides whose sequences coincide withthe C-terminal region of IL-16.

BACKGROUND OF THE INVENTION

Interleukin-16 (IL-16), previously named lymphocyte chemoattractantfactor (or LCF), is a pro-inflammatory lymphokine with chemoattractantactivity for resting CD4⁺ T lymphocytes. Subsequent studies indicatethat IL-16 activates signal transduction in CD4⁺ target cells includingmonocytes, eosinophils and pro-B cells, and stimulates a variety ofbiological activities in addition to chemotaxis. Among these activitiesare inhibition of retroviral replication (Maciaszek, et al., J. Immunol.158:5, 1997; Zhou, et al., Nature Medicine 3:659, 1997 and Baier, etal., Nature 378:563, 1995), upregulation of IL-2R and synergy with IL-2for CD4⁺ T cell proliferation (Parada, et al., J. Immunol. 160:2115,1998), induction of RAG-1 and RAG-2 expression in CD4⁺ pro-B cells(Szabo, et al., J. Immunol., 161:2248, 1998), and transient inhibitionof Mixed Lymphocyte Reaction (MLR)(Theodore, et al., J. Immunol.157:1958, 1996). Investigation of certain human diseases andexperimental murine models indicates that IL-16 participates ininflammatory conditions characterized by tissue recruitment of CD4⁺ Tlymphocytes and other CD4⁺ cell types. Conditions where IL-16 has beenidentified by ELISA and/or bioassay of body fluids, or byimmunohistochemical and in situ hybridization techniques, includebronchial asthma (Laherge et al., Am. J. Respir. Cell. Mol. Biol. 17:193, 1997), inflammatory bowel disease (Keates et al., Gastroenterology112, A110, 1997), Graves' disease (Cruikshank et al., J. Allergy Clin.Immunol. 99: 554, 1997), multiple sclerosis (Biddison et al., J.Immunol. 158: 3046, 1997) and bullous pemphigoid (Center et al., J.Invest. Dermatol. 81: 204, 1983). IL-16 is also implicated in thepathogenesis of rheumatoid arthritis (Klimiuk et al., J. Immunol. 162:4293-4299, 1999) and lupus (Lee et al., British J. Rheumatology 37:1334-1337, 1998). Thus, it would be desirable to identify and/orgenerate reagents capable of interfering with the IL-16 activity for thepurpose of treating inflammatory diseases.

The predicted amino acid sequence of IL-16 contains a central PDZmodule, and structural studies confirm that IL-16 assumes a corePDZ-like conformation with flexible N-terminal and C-terminal tails of17 and 14 residues, respectively (Muhlhahn et al., Nature StructuralBiology 5:682, 1998)). A synthetic oligopeptide corresponding to the 16C-terminal amino acids of human IL-16 (Arg106 to Ser121) has beenreported to inhibit the chemoattractant activity of natural andrecombinant human or murine IL-16 (Keane et al., J. Immunol. 160:5945,1998).

The present invention demonstrates that a series of peptidescorresponding to native or substituted sequences of the C-terminus ofIL-16 can inhibit IL-16 activity. Compositions and methods useful fortreating IL-16 mediated disorders are exploited using these peptides.

SUMMARY OF THE INVENTION

One embodiment of the present invention is directed to IL-16antagonists.

Another embodiment of the present invention is directed to IL-16antagonist peptides.

The IL-16 antagonist peptides of the present invention are at least 4amino acids in length and substantially correspond to the C-terminalsequence of human or murine IL-16 surrounding the Arg/Lys-Arg motif,i.e., R¹⁰⁶-R¹⁰⁷ of human IL-16, R¹⁰³-R¹⁰⁴ of murine IL-16 or K¹⁰⁶-R¹⁰⁷of IL-16 from squirrel monkey, for example.

A preferred IL-16 antagonist peptide of the present invention is thetetrameric peptide X_(aa0)RX_(aa1)X_(aa2) (SEQ ID NO:1), wherein X_(aa0)is Arg or Lys, and X_(aa1) and X_(aa2) can be any amino acid.Preferably, X_(aa1) and X_(aa2) are those amino acids found in thenative sequence of a mammalian IL-16, such as Lys or Thr for X_(aa1),and Ser for X_(aa2).

More preferably, X_(aa0)RX_(aa1)X_(aa2) is a tetramer having a sequencewhich coincides with the native sequence of a mammalian IL-16, e.g.,RRKS (SEQ ID NO:2), RRTS (SEQ ID NO:3), or KRKS (SEQ ID NO:4). Even morepreferably, such tetramer has Arg as the first amino acid. Homologs andanalogs of the tetramers of SEQ ID NO:2-4 are also contemplated by thepresent invention. For example, analogs of RRKS (SEQ ID NO:2) includeRRAS (SEQ ID NO:5) and RRKA (SEQ ID NO:6).

Another preferred IL-16 antagonist peptide of the present invention is atetrameric peptide having the sequence of X_(aa1)X_(aa2)X_(aa0)R (SEQ IDNO:8), wherein X_(aa0) is Arg or Lys, and X_(aa1) and X_(aa2) can be anyamino acid.

Preferably, X_(aa1) and X_(aa2) are those amino acids found in thenative sequence of a mammalian IL-16, e.g., Val for X_(aa1), and Ile orLeu for X_(aa2).

More preferably, X_(aa1)X_(aa2)X_(aa0)R is a tetramer having a sequencewhich coincides with the native sequence of a mammalian IL-16, such asVIRR (SEQ ID NO:9), VLRR (SEQ ID NO:10) and VIKR (SEQ ID NO:11). Evenmore preferably, such tetramer has Arg as the first amino acid. Homologsand analogs of these tetramers (SEQ ID NOS:9-11) are also contemplatedby the present invention.

Still another preferred IL-16 antagonist peptide of the presentinvention is a tetrameric peptide having the sequence ofX_(aa1)X_(aa0)RX_(aa2) (SEQ ID NO:12), wherein X_(aa0) is Arg or Lys,and X_(aa1) and X_(aa2) can be any one amino acid.

Preferably, X_(aa1) and X_(aa2) are those amino acids found in thenative sequence of a mammalian IL-16. For example, X_(aa1) can be Ile orLeu, and X_(aa2) can be Lys or Thr.

More preferably, X_(aa1)X_(aa2)X_(aa0)R is a tetramer having a sequencewhich coincides with the native sequence of an IL-16, such as IRRK (SEQID NO:13), IRRT (SEQ ID NO:14), LRRK (SEQ ID NO:15), and IKRK (SEQ IDNO:16). Even more preferably, such tetramer has Arg as the first aminoacid. Homologs and analogs of such tetramers are also contemplated bythe present invention.

Further according to the present invention, an IL-16 antagonist peptidecan be longer than a tetramer, as long as the such antagonist peptidecontains one of the tetrameric sequences described hereinabove, i.e.,X_(aa0)RX_(aa1)X_(aa2) (SEQ ID NO:1), X_(aa1)X_(aa0)RX_(aa2) (SEQ IDNO:8) or X_(aa1)X_(aa2)X_(aa0)R (SEQ ID NO:12), and as long as suchpeptide antagonizes at least one IL-16 biological activity.

Nucleic acid molecules coding for any of the above IL-16 antagonistpeptide of the present invention, expression vectors which include anyof such nucleic acid molecules, as well as related host cells containingsuch nucleotide sequences or vectors, are also contemplated by thepresent invention.

In a further aspect, the present invention provides antibodies directedagainst the IL-16 antagonist peptides of the present invention.

Preferably, the antibodies of the present invention are raised againstthose IL-16 antagonist peptides whose sequences coincide with thecorresponding sequences of a mammalian IL-16 protein, which antibodiescan antagonize or neutralize the activity of IL-16. Both polyclonalantibodies and monoclonal antibodies are contemplated by the presentinvention.

Functional derivatives of the monoclonal antibodies of the presentinvention are also contemplated, including Fab, Fab′, F(ab′)₂ of thepresent mAbs, single chain antibodies, humanized antibodies and thelike.

A related aspect of the present invention is directed to methods ofraising antibodies specific for the IL-16 antagonist peptides of thepresent invention by using such peptides as immunogens.

In another embodiment, the present invention provides pharmaceuticalcompositions which include one or more of the IL-16 antagonist peptidesor antibodies, and a pharmaceutically acceptable carrier. Thepharmaceutical compositions of the present invention can also includeother appropriate active ingredients, such as known anti-inflammatoryagents, e.g., anti-CD4 antibodies, anti-TNFα antibodies, NSAIDS,steroids, cyclosporin-A, or cytotoxic drugs.

Another aspect of the present invention provides methods of interferingwith, blocking or otherwise preventing the interaction or binding ofIL-16 with an IL-16 receptor by employing the IL-16 antagonistscontemplated by the present invention.

In a further aspect, the present invention provides methods of treatingan IL-16-mediated disorder in a subject by administering atherapeutically effective amount of a pharmaceutical composition of thepresent invention. In particular, IL-16-mediated disorders which can betreated by employing the methods of the present invention includeasthma, rheumatoid arthritis, inflammatory bowel disease, Graves'disease, multiple sclerosis, lupus and bullous pemphigoid.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 graphically depicts the structure of IL-16 and peptides used forinhibition studies. Mature IL-16 (released from pro-IL-16 by caspase-3cleavage) is a 121 amino acid polypeptide consisting of a centralPDZ-like domain flanked by N-terminal and C-terminal tails(crosshatched) of 17 and 14 residues, respectively. The arginineresidues at position 106 and 107 are within the boundary of the PDZdomain. The native terminal sequences are indicated below the cartoon.Oligopeptides corresponding to indicated C-terminal sequences fromArg¹⁰⁶ to Ser¹²¹ were prepared. Peptides made with alanine substitutionsof the native sequence are indicated in boldface.

FIG. 2 depicts the inhibition of IL-16-stimulated T cell motility byC-terminal peptides in chemotaxis assays using human T lymphocytes inthe presence or absence of C-terminal peptides. A, IL-16 inhibition byoligopeptides corresponding to native IL-16 sequences. Cell migration inresponse to rIL-16 at concentrations of 10⁻⁹ M (solid bar), 10⁻¹⁰ M(empty bar) and 10⁻¹¹ M (crosshatched bar) with or without peptides wascompared to cell migration in response to control buffer (considered as100%). Each of the indicated peptides was added at 10 μg/ml. Tenhigh-power fields were counted and the mean obtained for each condition.Results are expressed as the mean % control migration. SEM for threeexperiments. Comparisons between control and experimental conditionswere analyzed by Student=s t test; the asterisk indicates statisticalsignificance (P<0.05) for a difference in T cell migration at theindicated IL-16 concentration in the presence or absence of peptide. B,IL-16 inhibition by oligopeptides with alanine substitutions. Resultsare expressed as the mean % control migration SEM for four experiments.

FIG. 3 depicts specific inhibition of IL-16 by peptides. T cells werestimulated with gp120 or Leu 3a at 0.5 μg/ml (solid bars), 1.0 μg/ml(empty bars) and 5.0 μg/ml (crosshatched bars) in the presence orabsence of peptide RRKS (10 μg/ml) as indicated. Results are expressedas the mean % control migration SEM for three experiments.

FIG. 4 graphically depicts composition of recombinant IL-16 mutantsgenerated by PCR mutatgenesis and produced in E. coli. The nativeN-terminal and C-terminal sequences are represented as crosshatched barsflanking the central PDZ-like core. Deletions of 12 or 16 C-terminalresidues and 12 or 22 N-terminal residues are shown in the figure.Mutants with single Alanine substitutions are also indicated.

FIG. 5 depicts chemoattractant activity of mutated rIL-16. A, C-terminaland N-terminal deletion mutations. Concentrations of rIL-16 testedincluded 10⁻⁸ M (solid bar), 10⁻⁹ M (empty bar), 10⁻¹⁰ M (crosshatchedbar) and 10⁻¹¹ M (stippled bar). B, Chemoattractant activity of IL-16constructs with C-terminal point mutations. The IL-16 point mutationsincluded Arg¹⁰⁶ plus Arg¹⁰⁷ to alanine (IAAK), Arg¹⁰⁷ to alanine (IRAK),or Arg¹⁰⁶ to alanine (IARK).

FIG. 6 depicts Western blot analysis of native and mutated rIL-16.Native rIL-16 and C-terminal IL-16 deletion mutant proteins wereresolved by SDS/PAGE and transferred to nitrocellulose byelectroblotting. Duplicate blots were probed with polyclonal rabbitanti-IL-16 (upper panel), or monoclonal anti-IL-16 (mAb 17. 1; lowerpanel), detected with HRP-conjugated secondary Ab, and visualized bychemiluminescence. C-4 deletion (lane 1), C-8 (lane 2), C-12 (lane 3),C-16 (lane 4), native rIL-16 (lane 5).

FIG. 7 depicts inhibition of the MLR by native rIL-16 or rIL-16 withC-terminal point mutations. Stimulator cells consisted of PBMCpre-treated with mitomycin C. Responder cells were T lymphocytesisolated from a different donor and incubated in control buffer (NoIL-16), or pre-treated with native or mutated rIL-16 at 10⁻⁸ M (blackbars), 10⁻⁹ M (empty bars), 10⁻¹⁰ M (crosshatched bars), or 10⁻¹¹ M(stippled bars). The IL-16 point mutations included Arg¹⁰⁶ plus Arg¹⁰⁷to alanine (IAAK), Arg¹⁰⁷ to alanine (IRAK), or Arg¹⁰⁶ to alanine(IARK). Cultures were pulsed with [³H]thymidine on day 5 and harvestedon day 6 for scintigraphy. Results are expressed as mean cpm (withbackground subtracted). SD for three experiments.

FIG. 8 depicts inhibition of MLR by IL-16 deletion mutants. Respondercells were pre-incubated in control buffer (No IL-16) or pre-treatedwith (10⁻⁸ M to 10⁻¹¹ M) native rIL-16 or with the rIL-16 deletionconstructs C-12, C-16, N-12, N-22, C-16 plus N-12, or C-16 plus N-22.Asterisks indicate a significant difference (P<0.05) in mean cpmcomparing cells pre-treated with native rIL-16 or mutated rIL-16 at theidentical concentration.

FIG. 9 depicts the IL-16 sequences from various species. The IL-16sequences from African green monkey, rhesus monkey and mangeby areidentical. The Arg/Lys-Arg motif is underlined.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been found that aseries of peptides having sequences that substantially correspond tospecific regions of the C-terminus of IL-16 can inhibit the activity ofIL-16. Surprisingly, the present inventors have found that suchIL-16-inhibiting peptides can be as short as 4 amino acids in length.

Accordingly, the present invention is directed to IL-16 antagonists. By“IL-16 antagonist” is meant any molecule that inhibits, suppresses orcauses the cessation of at least one IL-16-mediated biological activityby, e.g., interfering with, blocking or otherwise preventing theinteraction or binding of IL-16 to an IL-16 receptor, e.g., the CD4receptor.

“An IL-16-mediated biological activity” as used herein includeschemotaxis of CD4+ cells such as CD4+ T cells, inhibition of retroviralreplication (such as inhibition of HIV and SIV in infected PBMCs),upregulation of IL-2R on CD4+ T cells, synergy with IL-2 for CD4⁺ T cellproliferation, induction of RAG-1 and RAG-2 expression in CD4⁺ pro-Bcells, and inhibition of Mixed Lymphocyte Reaction (MLR). These IL-16mediated biological activities can be determined using the assaysdescribed by Cruikshank et al. (Proc. Natl. Acad. Sci. USA 91:5109-5113, 1994); Maciaszek et al. (J. Immunol. 158:5, 1997), Zhou, etal. (Nature Medicine 3:659, 1997) and Baier et al. (Nature 378:563,1995); Parada et al. (J. Immunol. 160:2115, 1998); Szabo et al. (J.Immunol., 161:2248, 1998); and Theodore et al. (J. Immunol. 157:1958,1996), respectively. The teachings of these references are incorporatedherein by reference.

An IL-16 antagonist functions in two ways. The antagonist can competewith IL-16 for the cell surface receptor thereby interfering with,blocking or otherwise preventing the binding of IL-16 to an IL-16receptor. This type of antagonist, i.e., which binds the receptor butdoes not trigger signal transduction, is also referred to herein as a“competitive antagonist” and is a feature of the present invention.Alternatively, an IL-16 antagonist can bind to or sequester IL-16 withsufficient affinity and specificity to substantially interfere with,block or otherwise prevent binding of IL-16 to an IL-16 receptor,thereby inhibiting, suppressing or causing the cessation of at least oneIL-16-mediated biological activity, such as T-cell chemotaxis, forexample. This type of IL-16 antagonist, also termed a “sequesteringantagonist” is more specifically described in U.S. Pat. No. 6,723,697,issued from Ser. No. 09/929,922, filed on Aug. 15, 2001, which is adivisional of Ser. No. 09/368,632, filed on Aug. 5, 1999 and entitled“IL-16 Antagonists”, now abandoned, the teachings of which areincorporated herein by reference.

According to the present invention, preferred IL-16 antagonists includepeptides (referred to herein as “IL-16 antagonist peptides”) andantibodies.

According to the present invention, an IL-16 antagonist peptide is atleast 4 amino acids in length and substantially corresponds to theC-terminal sequence of human or murine IL-16 surrounding the Arg/Lys-Argmotif, i.e., R¹⁰⁶-R¹⁰⁷ of human IL-16, R¹⁰³-R¹⁰⁴ of murine IL-16 orK¹⁶⁰-R¹⁰⁷ of IL-16 from squirrel monkey and Aotus trivirgatus. Thenumbering of the amino acids are defined in accordance with thesequences of the mature, secreted form of IL-16. The sequences of themature IL-16 from human and mouse have been described by Keane et al.(J. Immunol 160: 5945-5954, 1998). See also FIG. 9. The sequences of thefull-length pro-IL-16 from African green monkey, rhesus monkey, mangeby,zebu, macaque, squirrel monkey and Aotus trivirgatus have been publishedby the Genbank database. The predicted sequences of the mature IL-16from these species are also included in FIG. 9.

By “substantially corresponds to” is meant peptides having sequencesthat are identical to the native sequences of the C-terminal region ofhuman or murine IL-16 surrounding the Arg/Lys-Arg motif, as well ashomologs and analogs of such peptides.

By “homologs” is meant the corresponding peptides from IL-16 proteins ofother mammalian species substantially homologous at the overall protein(i.e., mature protein) level to human or murine IL-16, so long as suchhomolog peptides retain the IL-16 antagonist property. The mammalianspecies can include African green monkey, rhesus monkey, mangeby, zebu,macaque, squirrel monkey and Aotus trivirgatus. According to the presentinvention, the IL-16 sequences from African green monkey, rhesus monkeyand mangeby are identical and share about 95% homology with human IL-16,and about 82.1% homology with murine IL-16, respectively.

By “substantial homologous” is meant the degree of amino acid homologyof at least about 65%, preferably at least about 70%, and morepreferably at least about 75%, which degree is the similarity indexcalculated using the Lipman-Pearson Protein Alignment program with thefollowing choice of parameters: Ktuple=2, Gap Penalty=4, and Gap LengthPenalty=12.

According to the present invention, the IL-16 antagonist peptides of thepresent invention antagonize human and murine IL-16 as well as IL-16molecules of other mammalian species that are substantially homologousto human or murine IL-16 proteins.

By “analogs” is meant peptides which differ by one or more amino acidalterations, which alterations, e.g., substitutions, additions ordeletions of amino acid residues, do not abolish the IL-16 antagonistproperties of the relevant peptides.

Thus, an analog of a peptide can have one or more amino acid residues ofthe peptide substituted, conservatively or non-conservatively. Examplesof conservative substitutions include the substitution of a non-polar(hydrophobic) residue such as I, V, L or M for another; the substitutionof one polar (hydrophilic) residue for another polar residue, such as Rfor K, Q for N, G for S, or vice versa; and the substitution of a basicresidue such as K, R or H for another or the substitution of one acidicresidue such as D or E for another. Examples of non-conservativesubstitutions include the substitution of a non-polar (hydrophobic)residue such as I, V, L, A, M for a polar (hydrophilic) residue such asC, Q, D, K and/or vice versa.

The phrase “analog” also includes the use of chemically derivatizedresidues in place of a non-derivatized residue as long as the peptideretains the requisite IL-16 antagonist properties.

Analogs also include addition of amino acids to the N-terminus orC-terminus of a relevant peptide. For example, the addition of cysteineto the N- or C-terminus of a peptide, by which, if desired, the peptidecan be covalently attached to a carrier protein, e.g., albumin. Suchattachment, it is believed, can minimize clearing of the peptide fromthe blood and also prevent proteolysis of the peptides.

In addition, for purposes of the present invention, peptides containingD-amino acids in place of L-amino acids are also included in the term“analogs”. The presence of such D-isomers may help minimize proteolyticactivity and clearing of the peptide.

A preferred IL-16 antagonist peptide of the present invention is atetrameric peptide having the sequence of X_(aa0)RX_(aa1)X_(aa2) (SEQ IDNO:1), wherein X_(aa0) is Arg or Lys, and X_(aa1) and X_(aa2) can be anyamino acid, which includes

A=Ala=Alanine,

R=Arg=Arginine,

N=Asn=Asparagine,

D=Asp=Aspartic acid,

C=Cys=Cysteine,

Q=Gln=Glutamine,

E=Glu=Glutamic acid,

G=Gly=Glycine,

H=His=Histidine,

I=Ile=Isoleucine,

L=Leu=Leucine,

K=Lys=Lysine,

M=Met=Methionine,

F=Phe=Phenylalanine,

P=Pro=Proline,

S=Ser=Serine,

T=Thr=Threonine,

W=Trp=Tryptophan,

Y=Tyr=Tyrosine and

V=Val=Valine.

Preferably, X_(aa1) and X_(aa2) are those amino acids found in thenative sequence of a mammalian IL-16. For example, X_(aa1) can be Lys(human, African green monkey, rhesus monkey, mangeby, zebu, macaque,squirrel monkey and Aotus trivirgatus) or Thr (murine), and X_(aa2) canbe Ser (human, African green monkey, rhesus monkey, mangeby, zebu,macaque, squirrel monkey, Aotus trivirgatus and murine).

More preferably, X_(aa0)RX_(aa1)X_(aa2) is a tetramer having a sequencewhich coincides with the native sequence of a mammalian IL-16. Examplesof such tetrameric sequences include RRKS (SEQ ID NO:2) (human, Africangreen monkey, rhesus monkey, mangeby, zebu and macaque), RRTS (SEQ IDNO:3) (murine), and KRKS (SEQ ID NO:4) (squirrel monkey and Aotustrivirgatus).

Even more preferably, X_(aa0)RX_(aa1)X_(aa2) coincides with the nativesequence of a mammalian IL-16 and X_(aa0) is Arg, for example, RRKS (SEQID NO:2) and RRTS (SEQ ID NO:3).

Homologs and analogs of any of these tetramers SEQ ID NOS:2-4 are alsocontemplated by the present invention. For example, analogs of tetramerRRKS (SEQ ID NO:2) of the present invention include RRAS (SEQ ID NO:5)and RRKA (SEQ ID NO:6), and an analog of RRTS (SEQ ID NO:3) is RRAS (SEQID NO:5) and RRTA (SEQ ID NO:7).

Another preferred IL-16 antagonist peptide of the present invention is atetrameric peptide having the sequence of X_(aa1)X_(aa2)X_(aa0)R (SEQ IDNO:12), wherein X_(aa0) is Arg or Lys, and X_(aa1) and X_(aa2) can beany amino acid.

Preferably, X_(aa1) and X_(aa2) are those amino acids found in thenative sequence of a mammalian IL-16. For example, X_(aa1) can be Val(human, African green monkey, rhesus monkey, mangeby, zebu, macaque,squirrel monkey, Aotus trivirgatus and murine), and X_(aa2) can Ile(human, African green monkey, rhesus monkey, mangeby, macaque, squirrelmonkey, Aotus trivirgatus and murine), or Leu (zebu).

More preferably, X_(aa1)X_(aa2)X_(aa0)R (SEQ ID NO:8) coincides with thenative sequence of a mammalian IL-16. Examples of such tetramericsequences include VIRR (SEQ ID NO:9) (human, African green monkey,rhesus monkey, mangeby, macaque and murine), VLRR (SEQ ID NO:10)(Zebu),and VIKR (SEQ ID NO:11) (squirrel monkey and Aotus trivirgatus).

Even more preferably, X_(aa1)X_(aa2)X_(aa0)R coincides with the nativesequence of a mammalian IL-16 and X_(aa0) is Arg, for example, VIRR (SEQID NO:9) and VLRR (SEQ ID NO:10).

Homologs and analogs of these tetramers (SEQ ID NOS: 9-11) are alsocontemplated by the present invention.

Still another preferred IL-16 antagonist peptide of the presentinvention is a tetrameric peptide having the sequence ofX_(aa1)X_(aa0)RX_(aa2) (SEQ ID NO:12), wherein X_(aa0) is Arg or Lys,and X_(aa1) and X_(aa2) can be any amino acid.

Preferably, X_(aa1) and X_(aa2) are those amino acids found in thenative sequence of a mammalian IL-16. For example, X_(aa1) can be Ile(human, African green monkey, rhesus monkey, mangeby, macaque, squirrelmonkey, Aotus trivirgatus and murine) or Leu (zebu), and X_(aa2) can beLys (human, African green monkey, rhesus monkey, mangeby, macaque, zebu,squirrel monkey and Aotus trivirgatus) or Thr (murine).

More preferably, X_(aa1)X_(aa2)X_(aa0)R (SEQ ID NO:12) coincides withthe native sequence of an IL-16. Examples of such tetrameric sequencesinclude IRRK (SEQ ID NO:13)(human, African green monkey, rhesus monkey,mangeby and macaque), LRRK (SEQ ID NO:15)(zebu), IKRK (SEQ IDNO:16)(squirrel monkey and Aotus trivirgatus), and IRRT (SEQ IDNO:14)(murine).

Even more preferably, X_(aa1)X_(aa0)RX_(aa2) (SEQ ID NO:12) coincideswith the native sequence of a mammalian L-16 and X_(aa0) is Arg, forexample, IRRK (SEQ ID NO:13), LRRK (SEQ ID NO:15) and IRRT (SEQ IDNO:14).

Homologs and analogs of such tetramers (SEQ ID NOS:13-16) are alsocontemplated by the present invention.

Further according to the present invention, an IL-16 antagonist peptidecan be longer than a tetramer, as long as such antagonist peptidecontains one of the tetrameric sequences described hereinabove, i.e.,X_(aa0)RX_(aa1)X_(aa2) (SEQ ID NO:1), X_(aa1)X_(aa0)RX_(aa2) (SEQ IDNO:12) or X_(aa1)X_(aa2)X_(aa0)R (SEQ ID NO:8), and as long as suchpeptide antagonizes at least one IL-16 biological activity. Preferably,the peptides contain X_(aa0)RX_(aa1)X_(aa2) (SEQ ID NO:1), morepreferably, X_(aa0) is Arg in SEQ ID NO:1. Generally speaking, thepeptide has less than 35 amino acids, preferably less than 25 aminoacids, more preferably less than 16 amino acids. The peptides of thepresent invention does not include RRKSLQSKETTAAGDS (SEQ ID NO:33).

Preferred antagonist peptides include those having sequences whichcoincide with the native C-terminal sequence of an IL-16 starting fromthe residue Arg/Lys, which is Arg¹⁰⁶ for human IL-16, or thecorresponding positions of other mammalian IL-16 molecules. Examples ofsuch peptides include 6-mers RRKSLQ (SEQ ID NO:17), RRTSLQ (SEQ IDNO:18), RRKSCM (SEQ ID NO:19), KRKSMQ (SEQ ID NO:20), 8-mers RRKSLQSK(SEQ ID NO:24), RRTSLQCK (SEQ ID NO:25), RRKSLQPK (SEQ ID NO:26),RRKSCMSK (SEQ ID NO:27), and KRKSMQSK (SEQ ID NO:28). Preferred peptidesinclude RRKSLQ (SEQ ID NO:17), RRTSLQ (SEQ ID NO:18), RRKSCM (SEQ IDNO:19), RRKSLQSK (SEQ ID NO:24), RRTSLQCK (SEQ ID NO:25), RRKSLQPK (SEQID NO:26), and RRKSCMSK (SEQ ID NO:27). Homologs and analogs of any ofthese tetramers are also contemplated by the present invention.

The EL-16 antagonist peptides of the present invention can be made by avariety of well known techniques. For example, the peptides can bechemically synthesized using standard solid-phase synthetic techniques,initially described by Merrifield in J. Am. Chem. Soc. 85:2149-2154(1963), or the solution methods as described in The Proteins, Vol. II.3d Ed., Neurath, H. et al., Eds., p. 105-237, Academic Press, New York,N.Y. (1976). See also Bodanszky, et al. Peptide Synthesis, John Wiley &Sons, 2d Ed., (1976); and J. Stuart and J. D. Young, Solid Phase PeptideSynthesis, Pierce Chemical Company, Rockford, Ill., (1984). Appropriateprotective groups for use in different peptide syntheses are describedin the above-mentioned texts as well as in J. F. W. McOmie, ProtectiveGroups in Organic Chemistry, Plenum Press, New York, N.Y. (1973).

Additionally, the peptides of the present invention can also be preparedby recombinant DNA techniques. Nucleotide sequences coding for peptidesof the present invention can be readily made by those skilled in the artand then inserted into an expression vector for producing the subjectpeptide in an appropriate host cell. Recombinantly produced peptides canbe purified following routine procedures.

Nucleic acid molecules coding for an IL-16 antagonist peptide of thepresent invention, and expression vectors which include any of suchnucleic acid molecules, as well as related host cells containing suchnucleotide sequences or vectors, are also contemplated by the presentinvention.

In a further aspect, the invention provides antibodies raised againstthe IL-16 antagonist peptides of the present invention. The antibodiesof the present invention do not include mAb14.1 or mAb 17.1 (see,Cruikshank et al., Proc. Natl. Acad. Sci. USA 91(11):5109-5113, 1994Hessel et al., J. Immunol. 160: 2998-3005, 1998 and Keane et al., J.Immunol. 160: 5945-5954, 1998).

Preferably, the antibodies of the present invention are raised againstthose IL-16 antagonist peptides whose sequences coincide with thecorresponding sequences of a mammalian IL-16 protein, preferably, humanIL-16. According to the present invention, such antibodies can alsoinhibit the IL-16 function by binding to the peptide epitopes of anIL-16 molecule required for interacting with an IL-16 receptor, therebyblocking and neutralizing at least one IL-16-mediated biologicalactivity. The antibodies of the present invention do not include mAb14.1or mAb 17.1 (see, Hessel et al., J. Immunol. 160: 2998-3005, 1998, andKeane et al., J. Immunol. 160: 5945-5954, 1998).

The antibodies of the present invention can be generated by well-knownmethods. The peptides, in combination with Freund=s adjuvant, can beinjected into an appropriate animal such as rabbit, mice, cow, guineapig, rat, donkey and the like. The peptides can be coupled to a carrierpolypeptide, e.g., KLH, prior to immunization as described in Ausubel etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York.

Both polyclonal antibodies and monoclonal antibodies can be preparedusing the immunized animal. The procedure for making polyclonal andmonoclonal antibodies is well known in the art and can be found in,e.g., Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, ColdSpring Harbor Press, 1988. Polyclonal antibodies can be readily purifiedfrom the serum of the immunized animal using a number of well knownprotein purification procedures such as affinity chromatography.Monoclonal clonal antibodies can be prepared by following the standardhybridoma techniques (see e.g. Kohler et al., Nature 256:495, 1975).Briefly, the spleens of the immunized animal can be removed, and theirlymphocytes fused to an immortal cell line. The resulting hybridomas canbe screened initially by binding affinity to the relevant peptideantigen, which can be determined by various immuno assays such as ELISA.Hybridomas that produce monoclonal antibodies specific for the relevantpeptide antigen can be further screened for the ability of inhibiting atleast one IL-16 mediated biological activity, such as chemotaxis of CD4+T cells. Such IL-16-inhibiting antibodies are considered to be usefulantagonists in the invention.

Functional derivatives of the monoclonal antibodies of the presentinvention are also contemplated. “Functional derivatives” refer toantibody molecules or fragments thereof that are derived from theinstant monoclonal antibodies and that have retained the antigenspecificity of the instant monoclonal antibodies. Examples of functionalderivatives include Fab, Fab′, F(ab′)₂ of the present mAbs, single chainantibodies, humanized antibodies and the like.

A single-chain antibody (SAb) is created by fusing together the variabledomains of the heavy and light chains using a short peptide linker,thereby reconstituting an antigen binding site on a single molecule.Such single-chain antibody variable fragments (Fvs) can be fused to allor a portion of the constant domains of the heavy chain of animmunoglobulin molecule, if necessary. The use of sAb avoids thetechnical difficulties in the introduction of more than one geneconstruct into host cells. Single chain antibodies and methods for theirproduction are known in the art. See, e.g., Bedzyk et al. (1990) J.Biol. Chem., 265:18615; Chaudhary et al. (1990) Proc. Natl. Acad. Sci.,87:9491; U.S. Pat. No. 4,946,778 to Ladner et al.; and U.S. Pat. No.5,359,046 to Capon et al.

The monoclonal antibodies of the present invention can be humanized toreduce the immunogenicity for use in humans. For example, to humanize amonoclonal antibody raised in mice, one approach is to make mouse-humanchimeric antibodies having the original variable region of the murinemAb, joined to constant regions of a human immunoglobulin. Chimericantibodies and methods for their production are known in the art. See,e.g., Cabilly et al., European Patent Application 125023 (published Nov.14, 1984); Taniguchi et al., European patent Application 171496(published Feb. 19, 1985); Morrison et al., European Patent Application173494 (published Mar. 5, 1986); Neuberger et al., PCT Application WO86/01533, (published Mar. 13, 1986); Kudo et al., European PatentApplication 184187 (published Jun. 11, 1986); Robinson et al.,International Patent Publication #PCT/US86/02269 (published 7 May 1987);Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Sun et al.,Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better et al., Science240:1041-1043 (1988). These references are incorporated herein byreference. Generally, DNA segments encoding the H and L chainantigen-binding regions of the murine mAb can be cloned from themAb-producing hybridoma cells, which can then be joined to DNA segmentsencoding C_(H) and C_(L) regions of a human immunoglobulin,respectively, to produce murine-human chimeric immunoglobulin-encodinggenes. Humanized antibodies can be made using a second approach, i.e.,to construct a reshaped human antibody, which has been described in,e.g., Maeda et al., Hum. Antibod. Hybridomas 2: 124-134, 1991, andPadlan, Mol. Immunol. 28: 489-498, 1991.

A related aspect of the present invention is directed to methods ofgenerating antibodies specific for the IL-16 antagonist peptides of thepresent invention by using such peptides as immunogens.

In another embodiment of the present invention, one or more IL-16antagonists, e.g., IL-16 antagonist peptides or antibodies, are includedin pharmaceutical compositions. Such pharmaceutical compositions areused in the treatment of IL-16 mediated disorders, such as IL-16mediated inflammatory diseases.

The pharmaceutical compositions of the present invention can alsoinclude other appropriate active ingredients, such as knownanti-inflammatory agents, e.g., anti-CD4 antibodies, anti-TNFαantibodies, NSAIDS, steroids, cyclosporin-A or cytotoxic drugs.

According to the present invention, the pharmaceutical compositions alsoincludes a pharmaceutically acceptable carrier. As used herein, apharmaceutically acceptable carrier includes any and all solvents,dispersion media, isotonic agents and the like. Except insofar as anyconventional media, agent, diluent or carrier is detrimental to therecipient or to the therapeutic effectiveness of the active ingredientscontained therein, its use in practicing the methods of the presentinvention is appropriate. The carrier can be liquid, semi-solid, e.g.pastes, or solid carriers. Examples of carriers include oils, water,saline solutions, alcohol, sugar, gel, lipids, liposomes, resins, porousmatrices, binders, fillers, coatings, preservatives and the like, orcombinations thereof.

In accordance with the present invention, the active ingredients of thepresent pharmaceutical compositions can be combined with the carrier inany convenient and practical manner, e.g., by admixture, solution,suspension, emulsification, encapsulation, absorption and the like, andcan be made in formulations such as tablets, capsules, powder, syrup,suspensions that are suitable for injections, implantations,inhalations, ingestions or the like. When appropriate, thepharmaceutical compositions of the present invention should be madesterile by well known procedures. For example, solutions can be madesterile by filter sterilization or autoclave. To obtain a sterilepowder, sterilized solutions can be vacuum-dried or freeze-dried asnecessary.

Another embodiment of the present invention provides methods ofinterfering with, blocking or otherwise preventing the interaction orbinding of IL-16 with an IL-16 receptor by employing the IL-16antagonists contemplated by the present invention.

In a further aspect of the present invention, the pharmaceuticalcompositions of the present invention are employed for the treatment ofIL-16 mediated pathological disorders. Thus, the present inventionprovides methods of treating an IL-16-mediated disorder in a subject byadministering a therapeutically effective amount of a pharmaceuticalcomposition of the present invention.

By “an IL-16-mediated disorder” is meant a pathological disorder, theonset, progression or the persistence of the symptoms of which requiresthe participation of IL-16 molecules. Particularly, IL-16-mediateddisorders contemplated by the present invention include asthma,rheumatoid arthritis, inflammatory bowel disease, Graves' disease,multiple sclerosis, lupus and bullous pemphigoid.

The term “treatment” refers to effective inhibition of the IL-16activity so as to prevent or delay the onset, retard the progression orameliorate the symptoms of the disorder.

The term “subject” refers to any mammalian subject. Preferably, thesubject is a human subject.

According to the present invention, for treating an IL-16-mediateddisorder in a mammalian subject, preferred pharmaceutical compositionsfor use are those constituted with IL-16 antagonist peptides orantibodies that effectively antagonize the function of the IL-16molecule of such mammalian species.

The term “therapeutically effective amount” means the dose required toeffect an inhibition of the IL-16 activity so as to prevent or delay theonset, slow down the progression or ameliorate the symptoms of thedisorder.

Precise dosages depend on depends on the disease state or conditionbeing treated and other clinical factors, such as weight and conditionof the subject, the subject's response to the therapy, the type offormulations and the route of administration. The precise dosage to betherapeutically effective and non-detrimental can be determined by thoseskilled in the art. As a general rule, a suitable dose of apharmaceutical composition for the administration to adult humans rangesfrom about 0.001 mg to about 20 mg per kilogram of body weight, morepreferably, in the range of about 0.01 mg to about 5 mg per kilogram ofbody weight. The peptides should preferably be administered in an amountof at least about 50 mg per dose, more preferably in an amount up toabout 500 mg per dose. Since the peptide compositions of this inventionwill eventually be cleared from the bloodstream, re-administration ofthe compositions may be required. Alternatively, implantation orinjection of the peptides provided in a controlled release matrix can beemployed.

The pharmaceutical compositions of the present invention can beadministered to the subject in any practical and convenient manner. Theroutes of administration which can be employed include the oral,ophthalmic nasal, topical, transdermal, or parenteral (e.g.,intravenous, intraperitoneal, intradermal, subcutaneous orintramuscular). In addition, the pharmaceutical compositions can beintroduced into the body, by injection or by surgical implantation orattachment, proximate to a preselected tissue or organ site such that asignificant amount of an active substance is able to enter the site bydirect diffusion, and preferably, in a controlled release fashion.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. The terms and expressions which have been employed in thepresent disclosure are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof. It is to be understood that variousmodifications are possible within the scope of the invention. All thepublications mentioned in the present disclosure are incorporated hereinby reference.

EXAMPLE 1 Materials and Methods

Peptides

Synthetic oligopeptides corresponding to native or altered C-terminalIL-16 sequences were made at the commercial facilities of ResearchGenetics, Inc. (Atlanta, Ga.).

Cell Preparation

Human peripheral blood mononuclear cells (PBMC) were isolated asdescribed (Center, et al., J. Immunol. 128:256, 1982; Cruikshank, etal., J. Immunol. 128:2569, 1982 and Cruikshank, et al., J. Immunol.138:3817, 1987) from the blood of healthy volunteers by densitycentrifugation on Ficoll-Paque reagent (Pharmacia, Piscataway, N.J.).The mononuclear cell layer was washed with medium 199 (M. A.Bioproducts, Walkersville, Md.) supplemented with 0.4% bovine serumalbumin, 25 mM HEPES buffer, and 100 U/ml of penicillin and 100 μg/mlstreptomycin (M199-HPS). Samples were enriched for T lymphocytes bynylon wool adherence as described (Julius, et al., Eur. J. Immunol.3:645, 1973). The nonadherent cells were >95% CD3⁺ as determined by flowcytometry.

Recombinant Proteins

Recombinant human IL-16 corresponding to the 121 C-terminal aminobiologically active cytokine cleaved from natural pro-IL-16 was producedin E. coli as a polyhistidine fusion protein using the expression vectorpET-30 LIC (Novagen, Madison, Wis.). Following lysis of transformedbacteria, the protein was purified by metal chelation chromatography andthe N-terminal polyhistidine tag was removed by cleavage withenterokinase.

The native IL-16 expression vector (pET-30/L-16¹²¹) was used as atemplate for PCR mutagenesis to create four recombinant IL-16 (rIL-16)mutant constructs with progressive four amino acid deletions at theC-terminus (C-4 to C-16), as well as deletions of 12 or 22 N-terminalresidues. Two double deletion constructs lacking the first 12 or 22N-terminal residues as well as the last 16 C-terminal residues of IL-16were also produced. Point mutations in C-terminal residues of rIL-16were generated by site-directed mutagenesis using the Stratagene QuickChange Kit (Stratagene, La Jolla, Calif.) according to themanufacturer=s specifications. The point mutations included alaninesubstitution for Arg¹⁰⁶, Arg¹⁰⁷, and Arg¹⁰⁶ plus Arg¹⁰⁷.

Western Blot Analysis

Native and mutated rIL-16 proteins were subjected to electrophoresisthrough a 15% SDS-polyacrylamide gel, then electrophoreticallytransferred to nitrocellulose membranes. The membranes were probed witheither polyclonal rabbit anti-rIL-16 or a murine anti-rIL-16 mAbdesignated clone 17.1. Secondary horse radish peroxidase(HRP)-conjugated anti-immunoglobulins were used at a concentration of1:5000, and the signal was visualized by chemiluminescence (Pierce,Rockford, Ill.).

Chemotaxis Assay

Cell migration was measured using a modified Boyden chemotaxis chamberas described (Center, et al., J. Immunol. 128:256, 1982; Cruikshank, etal., J. Immunol. 128:2569, 1982, and Cruikshank, et al., J. Immunol.138:3817, 1987). Cells were suspended (5×10⁶ cells/ml) in M199-HPS andloaded into the upper wells, separated by an 8 μm pore sizenitrocellulose membrane from lower wells. The lower wells were loadedwith control buffer or experimental chemoattractant stimuli, with orwithout synthetic peptides. After a 4 h incubation at 37° C., themembranes were removed and stained with hematoxylin, dehydrated bysequential washes in ethanol and propanol, then washed in xylene toclarify the filter for cell counting by light microscopy. Cell migrationwas quantified by counting the number of cells in the filter that hadmoved beyond a depth of 50 μm in five separate fields in duplicate wellsfor all conditions. Cell counts were compared with unstimulated controlcell migration which was normalized to 100%. Results are expressed asmean % control migration and the data analyzed for statisticalsignificance (P<0.05) by Student=s t test.

Mixed Lymphocyte Reaction

Stimulator cells for mixed lymphocyte reactions were prepared byincubating PBMC (10⁶/ml) with 25 μg/ml mitomycin C for 30 min. The cellswere then washed four times with RPMI 1640 medium supplemented with 25mM HEPES buffer, 100 U/ml penicillin, and 100 μg/ml streptomycin (RPMI1640-HPS), then resuspended in RPMI 1640-HPS supplemented with 10% fetalbovine serum (complete medium) at 10⁶ cells/ml. Responder cells wereprepared from an unrelated donor, suspended in complete medium at 10⁶cells/ml, and pre-incubated (1 h, 37° C.) with control buffer, or withrIL-16 or mutated rIL-16 constructs (10⁻⁹ M to 10⁻¹¹ M). Stimulatorcells were then added (1:1) and the cell mixtures were transferred inquadruplicate to 96-well round-bottom plates. Cell cultures were pulsedwith [³H] thymidine on day 5, harvested with a Titertek cell harvester,and counted in a Becton Dickinson scintillation counter on day 6.Results are expressed as mean % cpm above background. SEM. Data wereanalyzed for statistical significance (P<0.05) by Student=s t test.

EXAMPLE 2 Inhibition of IL-16-Stimulated T Lymphocyte Motility byC-terminal Oligopeptides

A series of oligopeptides derived from the 16 C-terminal of human IL-16residues were prepared (FIG. 1) and tested for their ability tostimulate the T lymphocyte motility using a modified Boyden chemotaxischamber assay.

Human T lymphocytes were loaded in the upper wells, and rIL-16 atconcentrations of 10⁻⁹ M, 10⁻¹⁰ M or 10⁻¹¹ M was loaded in the lowerwells, in the presence or absence of two 8-mer peptides corresponding toamino acids Arg¹⁰⁶ to Lys¹¹³ (SEQ ID NO:24), and Glu¹¹⁴ to Ser¹²¹ (SEQID NO:46) of IL-16. As shown in FIG. 2,A, only the Arg¹⁰⁶ to Lys¹¹³peptide (SEQ ID NO:24) inhibited IL-16 in this assay. The six-mer RRKSLQ(SEQ ID NO:17) also inhibited IL-16-stimulated T cell migration, but ascrambled peptide containing the same residues in a randomly chosensequence (SEQ ID NO:47) demonstrated no inhibitory activity. To furtherdefine the residues mediating inhibition, the eight residue sequencefrom Arg¹⁰⁶ to Lys¹¹³ was divided into RRKS (SEQ ID NO:2) and LQSK (SEQID NO:48). Only RRKS (SEQ ID NO:2) inhibited IL-16 chemoattractantactivity (FIG. 2,A).

The contribution of individual residues within RRKS was analyzed byalanine scanning (FIG. 2,B). Substitution of either Arg¹⁰⁶ or Arg¹⁰⁷ wasassociated with loss of inhibitory activity against IL-16-inducedchemotaxis. In contrast, the peptides RRAS (SEQ ID NO:5) and RRKA (SEQID NO:6) inhibited IL-16 as effectively as the native RRKS (SEQ IDNO:2).

To test whether inhibition by RRKS (SEQ ID NO:2) of chemotaxis inresponse to CD4 stimulation is specific for IL-16, peptide RRKS (SEQ IDNO:2) was tested in combination with two different CD4 ligands thatinduce T cell motility, HIV-1 gp120 (strain HIV-1_(3B)) and Leu 3a mAb.Cell migration in response to HIV-1 gp120 (13), or divalent anti-CD4 mAbLeu 3a was not blocked by this peptide (FIG. 3).

These data demonstrate that the four-residue peptide RRKS caneffectively and specifically inhibit the chemoattractant activity ofIL-16.

EXAMPLE 3 Chemoattractant Activity of IL-16 Deletion Mutants

IL-16 mutant constructs were created with progressive deletions of 4C-terminal amino acids from C-4 through C-16 (FIG. 4). The C-12construct terminates at Ser¹⁰⁸, retaining the RRKS motif. The C-16construct terminates at Ile¹⁰⁵, deleting RRKS and succeeding downstreamresidues. These mutant IL-16 molecules were tested in chemotaxis assays.As shown in FIG. 5, A, C-12 was active as native rIL-16, while the C-16deletion completely eliminated the chemoattractant activity. In similarexperiments, C-4 and C-8 deletion constructs demonstratedchemoattractant activities comparable to native rIL-16. These results,consistent with the peptide studies, indicate that residues within theRRKS motif are required for IL-16-stimulated chemoattractant activity.

To determine whether the N-terminal structures of Il-16 contribute tochemotactic signaling, two additional constructs with deletion of 12 or22 N-terminal amino acids were tested. Both the N-12 and N-22 deletionmutants demonstrated chemoattractant activity comparable to native IL-16(FIG. 5,A). These results indicate that the N-terminal domain is notrequired in receptor interactions activating cell motility.

EXAMPLE 4 Contribution of Specific C-terminal Residues to IL-16Chemoattractant Activity

To determine the contribution of individual residues within the RRKSmotif to chemoattractant signaling, and to test the activity of IL-16mutants with minimal structural alterations, a series of point mutationsusing alanine substitution were generated (FIG. 4). Replacement ofArg¹⁰⁷ alone, or Arg¹⁰⁶ plus Arg¹⁰⁷, completely abrogatedchemoattractant activity of the recombinant protein (FIG. 5,B). Incontrast, substitution of Arg¹⁰⁶ alone retained full activity. Theidentical pattern of motile responses was observed using a differentIL-16-responsive cell type, human peripheral blood monocytes.

To test whether multimer formation is disrupted by deletion or pointmutation, the above-generated mutant IL-16 molecules were assessed byHPLC. All of these constructs formed multimers similar to native IL-16.These observations indicate that mutation of Arg¹⁰⁷ directly interfereswith CD4 binding or activation by IL-16.

EXAMPLE 5 Western Analysis of IL-16 Mutant Proteins

Rabbit polyclonal anti-IL-16 Ab, as well as a murine monoclonalanti-IL-16 (clone 17.1) were generated using rIL-16 as an immunogen.This mAb was isolated by screening hybridoma supernatants forneutralization of IL-16 chemoattractant activity. Western blot analysiswas performed with native rIL-16 and the C-terminal deletion mutants(FIG. 6), using either the polyclonal Ab or the mAb for detection. Asexpected, the polyclonal Ab recognized native rIL-16 and all of thedeletion mutants. The mAb 17.1 detected native rIL-16 and the deletionmutants lacking 4, 8, or 12 C-terminal residues, as well as theN-terminal deletion mutants. However, mAb 17.1 failed to bind to C-16.The epitope for the neutralizing anti-IL-16 mAb 17.1 therefore maps tothe identical domain shown to be required for IL-16 chemoattractantactivity by peptide inhibition and mutation experiments.

EXAMPLE 6 Inhibition of the Mixed Lymphocyte Reaction

To determine whether other biological activities of IL-16 are mediatedby the C-terminal domain, the native and mutated rIL-16 constructs weretested for their capacity of inhibiting the one way MLR. Responder Tlymphocytes were pre-treated with rIL-16 or control buffer, thencultured with mitomycin C-treated stimulator PBMC from an unrelateddonor. Pre-treatment with 10⁻⁸ M native rIL-16 reduced thymidineincorporation on day 6 by nearly 70%, compared with untreated cells.Surprisingly, IL-16 mutants with the C-terminal point mutations whichlost chemoattractant activity retained full capacity to inhibit the MLR(FIG. 7). The C-16 deletion mutant was nearly as active as nativerIL-16, with a ˜1 log shift of the dose response (FIG. 8). Deletion of12 or 22 N-terminal residues resulted in a similar pattern as C-16; MLRinhibition was reduced but not eliminated. In contrast, constructs thatcombined the C-16 deletion with N-12 or N-22 lost all capacity toinhibit the MLR.

These data demonstrate that both N-terminal and C-terminal domains ofIL-16 are involved in receptor binding and activation, and that thestructural elements of IL-16 required for stimulating T cell motilityare different from those required for inhibition of Mixed LymphocyteReaction. TABLE I Seq ID Sequence Source 1 X_(aa0)RX_(aa1)X_(aa2) 2 RRKSh, ag, rm, mac, man, z 3 RRTS m 4 KRKS sm, at 5 RRAS analog 6 RRKAanalog 7 RRTA analog 8 X_(aa1)X_(aa2)X_(aa0)R 9 VIRR h, m, ag, rm, mac,man 10 VLRR z 11 VIKR sm, at 12 X_(aa1)X_(aa0)RX_(aa2) 13 IRRK h, ag,rm, mac, man 14 IRRT m 15 LRRK z 16 IKRK sm, at 17 RRKSLQ h, ag, rm,mac, man 18 RRTSLQ m 19 RRKSCM z 20 KRKSMQ sm, at 21 RRASLQ analog 22RRKALQ analog 23 RRTALQ analog 24 RRKSLQSK h 25 RRTSLQCK m 26 RRKSLQPKag, rm, man 27 RRKSCMSK z 28 KRKSMQSK sm, at 29 RRASLQSK analog 30RRKALQSK analog 31 RRTALQCK analog 32 RRASLQCK analog 33RRKSLQSKETTAAGDS h 34 RRTSLQCKQTTASADS m 35 RRASLQSKETTAAGDS analog 36RRKALQSKETTAAGDS analog 37 RRTALQCKQTTASADS analog 38 RRASLQCKQTTASADSanalog 39 Human IL-16 40 Murine IL-16 41 African green monkey IL-16 =rhesus monkey = mangeby 42 macaque IL-16 43 zebu IL-16 44 squirrelmonkey IL-16 45 Aotus trivirgatus IL-16 46 ETTAAGDS 47 RSQRLK 48 LQSKAbbreviations:h = human,m = murine,ag = African green monkey,rh = rhesus monkey,man = mangeby,z = zebu,mac = macaque,sm = squirrel monkey,at = Aotus trivirgatus

1. A pharmaceutical composition comprising an isolated Interleukin-16(IL-16) antagonist peptide and a pharmaceutically acceptable carrier,wherein said IL-16 antagonist peptide consists of a sequence selectedfrom the group consisting of RRKS (SEQ ID NO:2), RRTS (SEQ ID NO:3),KRKS (SEQ ID NO:4), RRAS (SEQ ID NO:5), RRKA (SEQ ID NO:6) and RRTA (SEQID NO:7).
 2. A pharmaceutical composition comprising an isolated IL-16antagonist peptide and a pharmaceutically acceptable carrier, whereinsaid IL-16 antagonist peptide consists of a sequence selected from thegroup consisting of RRKSLQ (SEQ ID NO:17), RRTSLQ (SEQ ID NO:18), RRKSCM(SEQ ID NO:19), KRKSMQ (SEQ ID NO:20), RRASLQ (SEQ ID NO:21), RRKALQ(SEQ ID NO:22) and RRTALQ (SEQ ID NO:23).
 3. A pharmaceuticalcomposition comprising an isolated IL-16 antagonist peptide and apharmaceutically acceptable carrier, wherein said IL-16 antagonistpeptide consists of a sequence selected from the group consisting ofRRKSLQSK (SEQ ID NO: 24), RRTSLQCK (SEQ ID NO:25), RRKSLQPK (SEQ IDNO:26), RRKSCMSK (SEQ ID NO:27), KRKSMQSK (SEQ ID NO:28), RRASLQSK (SEQID NO:29), RRKALQSK (SEQ ID NO:30), RRTALQCK (SEQ ID NO:31) and RRASLQCK(SEQ ID NO:32).
 4. A pharmaceutical composition comprising an isolatedIL-16 antagonist peptide and a pharmaceutically acceptable carrier,wherein said IL-16 antagonist peptide consists of a sequence selectedfrom the group consisting of RRTSLQCKQTTASADS (SEQ ID NO:34),RRASLQSKETTAAGDS (SEQ ID NO:35), RRKALQSKETTAAGDS (SEQ ID NO:36),RRTALQCKQTTASADS (SEQ ID NO:37) and RRASLQCKQTTASADS (SEQ ID NO:38). 5.A pharmaceutical composition comprising an isolated IL-16 antagonistpeptide and a pharmaceutically acceptable carrier, wherein said IL-16antagonist peptide consists of fewer than 16 amino acids and comprisesX_(aa0)RX_(aa1)X_(aa2) (SEQ ID NO: 1), wherein X_(aa0) is Arg or Lys,and X_(aa1) and X_(aa2) are any amino acids.
 6. The pharmaceuticalcomposition of claim 5, wherein X_(aa1) is selected from Lys, Thr, orAla; and X_(aa2) is selected from Serine or Ala.
 7. The pharmaceuticalcomposition of claim 5, wherein X_(aa0) is Arg.
 8. The pharmaceuticalcomposition of claim 7, wherein X_(aa1) is selected from Lys, Thr, orAla; and X_(aa2) is Ser or Ala.
 9. The pharmaceutical composition ofclaim 5, wherein X_(aa0) is Lys.
 10. The pharmaceutical composition ofclaim 9, wherein X_(aa1) is selected from Lys, Thr, or Ala; and X_(aa2)is Ser or Ala.
 11. A pharmaceutical composition comprising an isolatedIL-16 antagonist peptide and a pharmaceutically acceptable carrier,wherein said IL-16 antagonist peptide consists of fewer than 16 aminoacids and comprises a sequence selected from the group consisting ofRRKS (SEQ ID NO:2), RRTS (SEQ ID NO:3), KRKS (SEQ ID NO:4), RRAS (SEQ IDNO:5), RRKA (SEQ ID NO:6) and RRTA (SEQ ID NO:7).
 12. A pharmaceuticalcomposition comprising an isolated IL-16 antagonist peptide and apharmaceutically acceptable carrier, wherein said IL-16 antagonistpeptide consists of fewer than 16 amino acids and comprises the sequenceX_(aa1)X_(aa2)X_(aa0)R (SEQ ID NO:8), wherein X_(aa0) is Arg or Lys, andX_(aa1) and X_(aa2) are any amino acids.
 13. The pharmaceuticalcomposition of claim 12, wherein X_(aa1) is Val and X_(aa2) is Ile orLeu.
 14. The pharmaceutical composition of claim 12, wherein X_(aa0) isArg.
 15. The pharmaceutical composition of claim 14, wherein X_(aa1) isVal and X_(aa2) is Ile or Leu.
 16. The pharmaceutical composition ofclaim 12, wherein X_(aa0) is Lys.
 17. The pharmaceutical composition ofclaim 16, wherein X_(aa1) is Val and X_(aa2) is Leu or Ile.
 18. Apharmaceutical composition comprising an isolated IL-16 antagonistpeptide and a pharmaceutically acceptable carrier, wherein said IL-16antagonist peptide consists of fewer than 16 amino acids and comprises asequence selected from the group consisting of VIRR (SEQ ID NO:9), VLRR(SEQ ID NO:10) and VIKR (SEQ ID NO:11).
 19. A pharmaceutical compositioncomprising an isolated IL-16 antagonist peptide and a pharmaceuticallyacceptable carrier, wherein said IL-16 antagonist peptide consists offewer than 16 amino acids and comprises the sequenceX_(aa1)X_(aa0)RX_(aa2) (SEQ ID NO:12), wherein X_(aa0) is Arg or Lys,and X_(aa1) and X_(aa2) are any amino acids.
 20. The pharmaceuticalcomposition of claim 19, wherein X_(aa1) is Ile or Leu and X_(aa2) isLys, Thr or Ala.
 21. The pharmaceutical composition of claim 20, whereinX_(aa0) is Arg.
 22. The pharmaceutical composition of claim 21, whereinX_(aa1) is selected from Ile or Leu and X_(aa2) is Lys, Thr or Ala. 23.The pharmaceutical composition of claim 19, wherein X_(aa0) is Lys. 24.The pharmaceutical composition of claim 23, wherein X_(aa1) is selectedfrom Leu or Ile; and X_(aa2) is Lys, Thr or Ala.
 25. A pharmaceuticalcomposition comprising an isolated IL-16 antagonist peptide and apharmaceutically acceptable carrier, wherein said IL-16 antagonistpeptide consists of fewer than 16 amino acids and comprises a sequenceselected from the group consisting of IRRK (SEQ ID NO:13), IRRT (SEQ IDNO:14), LRRK (SEQ ID NO:15) and IKRK (SEQ ID NO:16).
 26. Apharmaceutical composition comprising an isolated IL-16 antagonistpeptide and a pharmaceutically acceptable carrier, wherein said IL-16antagonist peptide consists of fewer than 16 amino acids and comprises asequence selected from the group consisting of RRKSLQ (SEQ ID NO:17),RRTSLQ (SEQ ID NO:18), RRKSCM (SEQ ID NO:19), KRKSMQ (SEQ ID NO:20),RRASLQ (SEQ ID NO:21), RRKALQ (SEQ ID NO:22) and RRTALQ (SEQ ID NO:23).27. A pharmaceutical composition comprising an isolated IL-16 antagonistpeptide and a pharmaceutically acceptable carrier, wherein said IL-16antagonist peptide consists of fewer than 16 amino acids and comprises asequence selected from the group consisting of RRKSLQSK (SEQ ID NO:24),RRTSLQCK (SEQ ID NO:25), RRKSLQPK (SEQ ID NO:26), RRKSCMSK (SEQ IDNO:27), KRKSMQSK (SEQ ID NO:28), RRASLQSK (SEQ ID NO:29), RRKALQSK (SEQID NO:30), RRTALQCK (SEQ ID NO:31) and RRASLQCK (SEQ ID NO:32).
 28. Apharmaceutical composition comprising an isolated IL-16 antagonistpeptide and a pharmaceutically acceptable carrier, wherein said IL-16antagonist peptide consists of fewer than 25 amino acids and comprises asequence selected from the group consisting of RRTSLQCKQTTASADS (SEQ IDNO:34), RRASLQSKETTAAGDS (SEQ ID NO:35), RRKALQSKETTAAGDS (SEQ IDNO:36), RRTALQCKQTTASADS (SEQ ID NO:37) and RRASLQCKQTTASADS (SEQ IDNO:38).
 29. An isolated nucleic acid molecule coding for an IL-16antagonist peptide consisting of a sequence selected from the groupconsisting of RRTSLQCKQTTASADS (SEQ ID NO:34), RRASLQSKETTAAGDS (SEQ IDNO:35), RRKALQSKETTAAGDS (SEQ ID NO:36), RRTALQCKQTTASADS (SEQ ID NO:37)and RRASLQCKQTTASADS (SEQ ID NO:38).
 30. An antibody raised against anIL-16 antagonist peptide consisting of a sequence selected from thegroup consisting of RRTSLQCKQTTASADS (SEQ ID NO:34), RRASLQSKETTAAGDS(SEQ ID NO:35), RRKALQSKETTAAGDS (SEQ ID NO:36), RRTALQCKQTTASADS (SEQID NO:37) and RRASLQCKQTTASADS (SEQ ID NO:38).
 31. A method of treatingan IL-16 mediated disorder in a subject, comprising administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising an IL-16 antagonist peptide.
 32. The method ofclaim 31, wherein said IL-16 mediated disorder is an inflammatorydisease selected from asthma, rheumatoid arthritis, inflammatory boweldisease, Graves' disease, multiple sclerosis, lupus or bullouspemphigoid.
 33. The method of claim 32, further comprisingsimultaneously administering an anti-inflammatory agent selected from ananti-CD4 antibody, an anti-TNF antibody, NSAIDS, steroids, cyclosporin-Aor a cytotoxic drug.